Sensitive detection systems that are able to register the presence of very small target molecules and their interaction with other molecules are currently based on detection systems employing reagents such as dyes, fluorophores with or without quenchers, radioactive substances, etc. Most systems known in the art require the design of a fluorescent labelled hybridization probe which only emits a signal upon binding to its target nucleic acid. The following examples of various detection systems in the field of molecular biology are all well known and commonly used in the art:
The DNA binding dye technique utilizes that the amount of double stranded amplification product usually exceeds the amount of nucleic acid originally present in the sample to be analyzed. Double-stranded DNA specific dyes may be used, which upon excitation with an appropriate wavelength show enhanced fluorescence only if they are bound to double-stranded DNA. Preferably, only those dyes may be used which like SybrGreenl I, for example, do not affect the efficiency of the polymerase chain reaction (PCR).
The TaqMan probe is a single-stranded hybridization probe labelled with two components. When the first component is excited with light of a suitable wavelength, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer. During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is degraded by the 5′ exonuclease activity of the Taq polymerase during the subsequent elongation phase. As a result the excited fluorescent component and the quencher are spatially separated from one another and thus a fluorescence emission of the first component can be measured.
The Molecular Beacon is a hybridization probe that is also labelled with two components, a label and a quencher, the components preferably being located at each ends of the probe. As a result of the secondary structure of the probe, both components are in spatial vicinity in solution. After hybridization to the target nucleic acids both components are separated from one another such that after excitation with light of a suitable wavelength the fluorescence emission of the first component can be measured.
Single Label Probe (SLP) detection consists of a single oligonucleotide labelled with a single fluorescent dye at either the 5′- or 3′-end. Two different designs can be used for oligo labelling: G-Quenching Probes and Nitroindole-Dequenching probes. In the GQuenching embodiment, the fluorescent dye is attached to a C at oligo 5′- or 3′-end. Fluorescence decreases significantly when the probe is hybridized to the target, in case two G's are located on the target strand opposite to C and in position 1 aside of complementary oligonucleotide probe. In the Nitroindole Dequenching embodiment, the fluorescent dye is attached to Nitroindole at the 5′- or 3′-end of the oligonucleotide. Nitroindole somehow decreases the fluorescent signaling of the free probe. Fluorescence increases when the probe is hybridized to the target DNA due to a dequenching effect.
The FRET hybridization probe is especially useful for all kinds of homogenous hybridization assays. It is characterized by a pair of two single-stranded hybridization probes which are used simultaneously and are complementary to adjacent sites of the same strand of the amplified target nucleic acid. Both probes are labelled with different fluorescent components. When excited with light of a suitable wavelength, a first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer such that a fluorescence emission of the second component can be measured when both hybridization probes bind to adjacent positions of the target molecule to be detected. When annealed to the target sequence, the hybridization probes must sit very close to each other, in a head to tail arrangement. Usually, the gap between the labelled 3′ end of the first probe and the labelled 5′ end or the second probe is as small as possible, i.e. 1-5 bases. This allows for a close vicinity of the FRET donor compound and the FRET acceptor compound, which is typically 10-100 Angstroem.
In addition to and as an alternative it would be desirable to obtain a sensitive detection system devoid of the requirement for the currently used labels such as e.g. dyes, fluorophores with and without quenchers, radioactive substances, etc.